Kidney microstructural features at the time of donation predicts long-term risk of chronic kidney disease in living kidney donors

Abstract

Objective

To determine whether microstructural features on a kidney biopsy obtained during a kidney transplant surgery predict long-term risk of chronic kidney disease in the donor.

Patients and Methods

We studied kidney donors from May 1, 1999 through December 31, 2018 with a follow-up survey for the results of recent blood pressure and kidney function tests (estimated glomerular filtration rate [eGFR] and proteinuria). If not recently available, blood pressure and eGFR levels were requested from a local clinic. Microstructural features on kidney biopsy at the time of donation were assessed as predictors of hypertension and kidney function after adjusting for years of follow-up, baseline age, gender, and clinical predictors.

Results

There were 807 donors surveyed a mean 10.5 years after donation. An eGFR <45 mL/min/1.73 m² in 6.4% (43/673) of donors was predicted by larger glomerular volume per standard deviation (OR=1.48, 95%CI:1.08, 2.04) and nephron number below age-specific 5^th percentile (OR=3.38, 95%CI 1.31, 8.72). An eGFR <60 mL/min/1.73 m² in 42% (286/673) of donors was not predicted by any microstructural feature. Residual eGFR (post-donation/pre-donation eGFR) was predicted by nephron number below age-specific 5^th percentile (−6.07%, 95%CI: −10.24, −1.89). Self-reported proteinuria in 5.1% (40/786) of donors was predicted by larger glomerular volume (OR=1.42, 95%CI: 1.08, 1.86). Incident hypertension in 19% (119/633) of donors was not predicted by any microstructural features.

Conclusions

Low nephron number for age and larger glomeruli are important microstructural predictors for long-term risk of chronic kidney disease after living kidney donation.

1. INTRODUCTION

Living kidney donors undergo an exhaustive evaluation to ensure normal kidney function and to exclude risk factors for chronic kidney disease (CKD). Despite careful selection, a small subset of donors has low renal function post-donation,^1–3 and some recent studies suggest an increased risk of kidney failure or mortality compared to controls.^4–6 It has been also reported that specific donor populations, including African Americans, obese, hypertensive, or donors related to a recipient with end-stage renal disease (ESRD) have a higher risk of kidney failure.^7–11 There may be subclinical microstructural features in the kidneys of these living donors that contribute towards risk of chronic kidney disease after donation. The pre-donation computed tomography (CT) scan can detect kidney microstructural abnormalities, but there is often uncertainty regarding their clinical importance to donor selection.¹² A kidney biopsy may detect subclinical pathology, but is not routinely available prior to transplant. Thus, it is unclear whether the development of CKD in some living kidney donors is only related to events that occur after donation, or is due to some extent, to pathology already present at the time of donation.¹³

The Aging Kidney Anatomy study has characterized microstructural kidney biopsy features of the living donor kidney at the time of kidney implantation. Microstructural features can be classified into measures of nephron size, nephron number, or nephrosclerosis and associate with age, kidney function, and CKD risk factors of the donor, as well as with the macrostructure of the kidney.^14–17 They also modestly predict a lower post-donation glomerular filtration rate (GFR), albuminuria, and hypertension early after donation,¹⁸ and the death-censored risk of graft failure in the kidney recipient.¹⁹

Long-term follow-up is needed to more fully comprehend the clinical importance of baseline microstructural features in the kidney at the time of donation. Thus, we performed a cohort study from the living donors in the Aging Kidney Anatomy study to determine whether nephron size, nephron number per kidney, or nephrosclerosis at the time of donation predict a long-term risk of developing low GFR, self-reported proteinuria, or hypertension.

2. METHODS

2.1. Study design and study sample

This is a prospective cohort study of living kidney donors (18 years or older) at the Mayo Clinic sites in Minnesota and Arizona from May 1, 1999 through December 31, 2018 in the Aging Kidney Anatomy Study.^{18, 19} Our inclusion criteria required a time-zero biopsy during the transplant surgery with ≥2 mm² of non-distorted cortex with at least 4 glomeruli. To study long-term kidney function outcomes, we targeted donors with 5 or more years of follow-up for participation. The Mayo Clinic Survey Research Center contacted these donors via mailed surveys and follow-up phone calls from April 1, 2017 to December 31, 2018 using Accurint™ (www.accurint.com) to find contact information. Donors were allowed to complete the survey via mail or phone interview. Donors were asked to provide recent (within past 2 years) blood pressure readings, height, weight, and serum creatinine levels along with the dates of testing. Those lacking a recent blood pressure, height, weight, or serum creatinine were offered remuneration to obtain these tests from a local provider. This study was completed under IRB approval with a signed consent form completed by each participant.

2.2. Clinical characteristics

All kidney donors underwent a thorough medical evaluation prior to donation that included a prescheduled series of tests as previously described.¹⁸ The predonation evaluation included serum creatinine to estimate GFR using the CKD-EPI equation,²⁰ urinary iothalamate clearance to measure GFR, 24-hour urine albumin quantification, body mass index (BMI), office blood pressure readings, and CT scan imaging of the kidneys obtained at the time of donor evaluation. Acceptance criteria for donation varied by site and era, but in general included 24-hour urine albumin excretion <30 mg and a measured GFR normal for age. Mild hypertension in older donors and moderate obesity (BMI 30 to 35 kg/m²; occasionally up to 40 kg/m² in older donors) were allowed. Hypertension was defined as a pre-existing diagnosis of hypertension, an office systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg, or use of antihypertensive medication(s) to treat hypertension. Acceptable pre-donation “mild” hypertension was defined by either 140–159/90–99 mm Hg or controlled with one antihypertensive medication (with or without a thiazide diuretic). Potential donors with more severe hypertension or with evidence for target organ damage were excluded from donation. Patients with diabetes mellitus or cardiovascular disease were not acceptable candidates. Related donors were defined by being a blood relative of the kidney recipient. Tobacco smoking status was identified from the medical records at the time of donor evaluation.

2.3. Microstructural features on biopsy

As part of routine clinical care, intraoperative needle core biopsy of the renal cortex was performed at the time of transplantation. The tissue specimen was fixed in formalin and embedded in paraffin. Two sections (2- to 3-μm thickness) from the biopsy core were stained, one with periodic acid–Schiff and one with Masson trichrome, and were subsequently scanned into high-resolution digital images (Aperio XT digital scanner; Leica Biosystems). Nephron size on biopsy was characterized by mean non-sclerotic glomerular volume, cortex volume per glomerulus (reciprocal of nonsclerotic glomerular volumetric density), and mean cross-sectional tubular area as previously described (Supplemental Figures 1 and 2).¹⁵ Nephrosclerosis on biopsy was characterized by the percentage of glomeruli that were globally sclerosed, the percentage interstitial fibrosis/tubular atrophy (IF/TA) of the cortex area, the number of distinct IF/TA foci, and the severity of arteriosclerosis.¹⁵ The severity of arteriosclerosis was determined by the percentage of luminal stenosis due to intimal thickening in the small-medium artery (if any present) most orthogonal to its axis. These were performed by personnel unaware of the donors’ characteristics and outcomes (Supplemental Figure 3). Presence of any arteriolar hyalinosis required review of all 12 biopsy section slides by a pathologist to be detected (data only available for Mayo Clinic Minnesota). The Supplemental Methods provides further details on measuring and calculating microstructural features from kidney biopsy images.

2.4. Nephron number

Predonation CT images from the angiogram/cortical phase were downloaded onto a workstation for processing. The kidney cortical volumes were segmented using a semi-automated algorithm (ITK-SNAP software, version 2.2; University of Pennsylvania, Philadelphia, PA) Supplemental Figure 4.¹⁵ Nephron number per kidney was calculated from the product of cortical volume and non-sclerotic glomerular volumetric density as previously described.¹⁶

2.5. Kidney function outcomes

Serum creatinine levels at baseline and at follow-up were converted to estimated GFR (eGFR) by the CKD-EPI equation.²⁰ Residual eGFR was calculated by follow-up eGFR divided by the pre-donation eGFR × 100%. We assessed for a follow-up eGFR <45 mL/min/1.73 m² as an outcome in addition to eGFR <60 mL/min/1.73 m²; this lower threshold is considered a more clinically significant definition of CKD after a nephrectomy.²¹ To assess the validity of self-reported serum creatinine, we correlated the self-reported serum creatinine to the temporally closest available electronic serum creatinine in the local donor subset with local laboratory testing (within the Mayo Clinic Health System). Hypertension after donation was identified by a diagnosis by a health care provider, systolic blood pressure (SBP) ≥140 mm Hg, diastolic blood pressure (DBP) ≥90 mm Hg, or use of antihypertensive medication(s) to treat hypertension. Proteinuria is often reported from urine dipsticks obtained during acute illnesses such as a urinary tract infection. We were concerned that requiring a standardized assessment of proteinuria among all participants would decrease participation. Instead, proteinuria was determined by a survey question “Has a care provider informed you about abnormal protein in your urine?”

2.6. Statistical analysis

Nephrosclerosis and nephron number were dichotomized at the abnormal 95^th (or 5^th) percentiles for age as previously identified (Supplemental Tables 1 and 2).¹⁸ Nephron size measures were analyzed as continuous variables. Each microstructural feature was evaluated for its association with each outcome. With a single follow-up survey, a time-to-event analysis was not possible. Instead, linear regression was used for predicting residual eGFR and logistic regression for predicting eGFR <45 or <60 mL/min/1.73 m², self-reported proteinuria, and hypertension at follow-up. The risk of hypertension was only assessed in donors who did not have baseline hypertension. Multivariate imputation by chained equations (MICE) was used to impute missing covariate values (Supplemental Table 3).²² Coefficients, ORs, 95% CIs, and P-values were generated by combing summaries of nine MICE-imputed data sets using the methods of Rubin²³ and Li et al.²⁴ Models were unadjusted, then adjusted for age, gender and follow-up time, or further adjusted for clinical characteristics that also predicted the outcome. Statistical analyses were performed using R version 3.4.2.

3. RESULTS

3.1. Donor characteristics

There were 807 living kidney donors studied (Figure 1), of which 673 had a follow-up serum creatinine level. The survey was completed a mean of 10.5 years postdonation. Baseline clinical and biopsy characteristics of the cohort are shown in Table 1. These donors who responded to the survey were more likely to be older, female, non-smoker, unrelated to the recipient, and have a lower eGFR and more nephrosclerosis on biopsy than non-responders (Supplemental Table 4). None of the donors reported renal replacement therapy during the follow-up period.

Figure 1.

Sampling of living kidney donors.

Table 1.

Baseline characteristics of the 807 donors studied.

Clinical Characteristics	Mean ± SD or n (%)
Age, years	47.0 ± 11.9
Male	297 (37%)
Body mass index, kg/m2	27.6 ± 4.9
Predonation hypertension	154 (19%)
Predonation systolic blood pressure, mm Hg	120.1 ± 15.0
Predonation diastolic blood pressure, mm Hg	72.9 ± 9.5
Smoker at donor evaluation	93 (12%)
eGFR, mL/min/1.73 m2	87.9 ± 14.9
Related to recipient a	405 (50%)
Race
white	736 (91%)
non-white	30 (4%)
unknown	41 (5%)
Nephron size and number
Glomerular volume, mm3	0.0026 ± 0.0011
Cortex volume per glomerulus, mm3	0.08 ± 0.04
Mean tubular cross-sectional area, μm2	4,471 ± 1,492
Nephron number per kidney	864,900 ± 382,800
Nephrosclerosis
Globally sclerotic glomeruli, %	3.8 ± 6.6
Number of IF/TA foci
0	599 (74%)
1	128 (16%)
2	25 (3%)
3	26 (3%)
4	14 (2%)
≥5	15 (2%)
Percentage IF/TA
0%	599 (74%)
<1%	63 (8%)
1%–5%	110 (14%)
6%–10%	27 (3%)
>10%	8 (1%)
Artery luminal stenosis, %	35.5 ± 23.4
Any arteriolar hyalinosis b	92 (14%)

^a.86.7% first degree, 7.4% second degree, and 5.9% third degree relative

^b.Assessed only in 659 kidney donors from Mayo Clinic Minnesota with 12 slides available to review.

SD = Standard Deviation

3.2. Predictors of hypertension

After excluding donors with baseline hypertension, 18.8% (n=119) developed hypertension during follow-up. The mean systolic blood pressure of those who did versus did not develop hypertension was 130 vs. 117 mm Hg (p<.001). Clinical predictors of developing hypertension were longer time since donation, male sex, higher baseline BMI, and higher baseline blood pressure (Table 2). Biopsy measures of larger nephron size predicted hypertension, but not after adjusting for BMI and baseline blood pressure. Nephron number and nephrosclerosis did not predict hypertension before or after adjusting for clinical characteristics (Table 2).

Table 2.

Predictors of incident hypertension that occurred in 119 of 633 donors (18.8%) without baseline hypertension.^c

	Unadjusted			Adjusted for age, gender, and follow up time			Further adjusted for significant clinical predictors a
Clinical Predictors	OR	95% CI	P	OR	95% CI	P	OR	95% CI	P
Time since donation, 5 year	1.57	(1.13, 2.18)	.007	-	-	-	-	-	-
Age, 10 years	1.06	(0.89, 1.26)	.51	-	-	-	-	-	-
Male	1.54	(1.03, 2.31)	.04	-	-	-	-	-	-
Body mass index, 5 kg/m2	1.33	(1.08, 1.64)	.007	1.29	(1.04, 1.59)	.02	-	-	-
Smoker at donor evaluation	1.22	(0.68, 2.17)	.51	1.18	(0.66, 2.12)	.58	-	-	-
Systolic blood pressure, 10 mmHg	1.64	(1.36, 1.97)	<.001	1.56	(1.27, 1.91)	<.001	-	-	-
Diastolic blood pressure, 10 mmHg	2.09	(1.59, 2.74)	<.001	2.05	(1.56, 2.69)	<.001	-	-	-
eGFR, 10 ml/min/1.73 m2	0.97	(0.85, 1.11)	.63	1.02	(0.87, 1.19)	.82	-	-	-
Related to recipient	1.34	(0.89, 2.00)	.16	1.27	(0.84, 1.93)	.25	-	-	-
Nephron size and number
Glomerular volume, standard deviation (SD)	1.29	(1.07, 1.55)	.009	1.22	(1.00, 1.48)	.05	1.10	(0.89, 1.36)	.37
Cortex volume per glomerulus, SD	1.28	(1.05, 1.55)	.01	1.22	(1.00, 1.49)	.05	1.16	(0.94, 1.43)	.16
Mean tubular cross-sectional area, SD	1.26	(1.03, 1.54)	.03	1.16	(0.94, 1.43)	.16	1.14	(0.91, 1.41)	.25
Nephron number <5th percentile b	1.60	(0.78, 3.30)	.20	1.62	(0.78, 3.36)	.20	1.59	(0.74, 3.40)	.23
Nephrosclerosis
Globally sclerotic glomeruli >95th percentile b	1.30	(0.57, 2.94)	.53	1.46	(0.64. 3.35)	.37	1.50	(0.64, 3.51)	.35
Percentage IF/TA >95th percentile b	0.64	(0.28, 1.45)	.28	0.63	(0.28, 1.45)	.28	0.59	(0.25, 1.40)	.23
Number of IF/TA foci >95th percentile b	1.40	(0.58, 3.36)	.45	1.55	(0.63, 3.79)	.33	1.43	(0.57, 3.59)	.45
Artery luminal stenosis >95th percentile b	1.18	(0.55, 2.52)	.68	1.17	(0.54, 2.52)	.70	1.15	(0.52, 2.54)	.73
Any arteriolar hyalinosis	1.47	(0.79, 2.72)	.22	1.22	(0.64, 2.33)	.54	1.18	(0.61, 2.30)	.62

^a.Adjusted for time from donation, age, gender, BMI, predonation office systolic blood pressure and diastolic blood pressure.

^b.Age specific thresholds in Supplemental Table 1 and Supplemental Table 2.

^cThere were 20 patients who did not answer the hypertension query on the follow-up survey.

3.3. Predictors of eGFR

There was good correlation (r = 0.85, 95% CI 0.77, 0.90) and no bias (0.01 mg/dl, p=.65) between the serum creatinine obtained from the survey and the serum creatinine in the medical record among the subset of 82 donors with local care (Supplemental Figure 5). The donors that reported a follow-up serum creatinine level on survey (n=673) were more likely to be older, female, and have a lower baseline eGFR compared to those that did not report a follow-up serum creatinine level (n=134) (Supplemental Table 5).

There were 43/673 (6.4%) donors who developed an eGFR <45 ml/min/1.73m². Clinical predictors of an eGFR <45 ml/min/1.73 m² were older age, hypertension, and lower baseline eGFR (Table 3). In unadjusted analysis, larger glomerular volume, larger cortex per glomerulus, and low nephron number for age predicted a post-donation eGFR <45 ml/min/1.73 m². After adjusting for clinical predictors, only larger glomerular volume and low nephron number for age predicted an eGFR <45 ml/min/1.73 m². There were 286/673 (42.5%) donors who developed an eGFR <60 ml/min/1.73m². Clinical predictors of an eGFR <60 ml/min/1.73 m² were less time since donation, older age, non-smoker, hypertension, higher blood pressure, and lower baseline eGFR (Table 4). In unadjusted analysis, IF/TA foci higher than expected for age predicted eGFR<60 ml/min/1.73 m². After adjusting for clinical predictors, no microstructural feature predicted eGFR <60 ml/min/1.73 m². The mean residual eGFR was 74.8%. Clinical predictors of a lower residual eGFR were shorter follow-up time and baseline older age, non-smoker, and higher eGFR (consistent with regression to the mean) (Table 5). In unadjusted analysis, microstructural predictors of lower residual eGFR were not evident, but after adjusting for clinical predictors, low nephron number for age predicted a lower residual eGFR (Table 5).

Table 3.

Predictors of eGFR< 45 ml/min/1.73 m² that occurred in 43 of 673 donors (6.4%) with follow-up serum creatinine levels.

	Unadjusted			Adjusted for age, gender, and follow up time			Further adjusted for significant clinical predictors a
Clinical Predictors	OR	95% CI	P	OR	95% CI	P	OR	95% CI	P
Time since donation, 5 year	0.79	(0.48, 1.31)	.36	-	-	-	-	-	-
Age, 10 years	1.93	(1.42, 2.63)	<.001	-	-	-	-	-	-
Male	1.65	(0.89, 3.08)	.11	-	-	-	-	-	-
Body mass index, 5 kg/m2	1.06	(0.78, 1.43)	.72	1.02	(0.72, 1.44)	.91	-	-	-
Smoker at donor evaluation	0.61	(0.18, 2.03)	.42	0.77	(0.22, 2.62)	.67	-	-	-
Hypertension	2.23	(1.15, 4.31)	.02	1.37	(0.67, 2.78)	.38	-	-	-
Systolic blood pressure, 10 mmHg	0.99	(0.81, 1.22)	.92	0.84	(0.67, 1.06)	.14	-	-	-
Diastolic blood pressure, 10 mmHg	1.01	(0.73, 1.40)	.96	0.89	(0.63, 1.26)	.50	-	-	-
eGFR, 10 ml/min/1.73 m2	0.44	(0.33, 0.58)	<.001	0.48	(0.35, 0.65)	<.001	-	-	-
Related to recipient	1.21	(0.65, 2.25)	.55	1.63	(0.86, 3.12)	.14	-	-	-
Nephron size and number
Glomerular volume, standard deviation (SD)	1.39	(1.06, 1.84)	.02	1.44	(1.07, 1.96)	.02	1.48	(1.08, 2.04)	.01
Cortex volume per glomerulus, SD	1.35	(1.06, 1.73)	.02	1.30	(1.00, 1.69)	.05	1.27	(0.97, 1.66)	.08
Mean tubular cross-sectional area, SD	1.32	(0.98, 1.77)	.06	1.28	(0.93, 1.76)	.14	1.21	(0.87, 1.69)	.26
Nephron number <5th percentile b	3.02	(1.26, 7.25)	.01	3.58	(1.42, 9.01)	.007	3.38	(1.31, 8.72)	.01
Nephrosclerosis
Globally sclerotic glomeruli >95th percentile b	1.64	(0.56, 4.86)	.37	1.85	(0.61, 5.68)	.28	1.47	(0.46, 4.70)	.52
Percentage IF/TA >95th percentile b	1.43	(0.54, 3.8)	.47	1.74	(0.63, 4.77)	.28	1.82	(0.64, 5.22)	.26
Number of IF/TA foci >95th percentile b	2.73	(1.00, 7.46)	.05	2.33	(0.79, 6.82)	.12	2.58	(0.81, 8.22)	.11
Artery luminal stenosis >95th percentile b	0.89	(0.17, 4.58)	.89	1.06	(0.21, 5.50)	.94	1.03	(0.20, 5.22)	.97
Any arteriolar hyalinosis	1.36	(0.54, 3.39)	.51	0.83	(0.31, 2.18)	.70	0.88	(0.33, 2.39)	.80

^a.Adjusted for time from donation, age, gender, and eGFR

^b.Age specific thresholds in Supplemental Table 1 and Supplemental Table 2

Table 4.

Predictors of eGFR< 60 ml/min/1.73 m² that occurred in 286 of 673 donors (42.5%) with follow-up serum creatinine levels.

	Unadjusted			Adjusted for age, gender, and follow up time			Further adjusted for significant clinical predictors a
Clinical Predictors	OR	95% CI	P	OR	95% CI	P	% diff.	95% CI	P
Time since donation, 5 year	0.71	(0.56, 0.92)	.008	- -	-	-	-	-
Age, 10 years	2.33	(1.97, 2.76)	<.001	- -	-	-	-	-
Male	1.01	(0.73, 1.39)	.96	- -	-	-	-	-
Body mass index, 5 kg/m2	1.11	(0.95, 1.29)	.18	1.12	(0.95, 1.33)	.18	-	-
Smoker at donor evaluation	0.41	(0.24, 0.72)	.002	0.50	(0.28, 0.92)	.02	-	-
Hypertension	2.25	(1.53, 3.30)	<.001	1.24	(0.81, 1.89)	.33	-	-
Systolic blood pressure, 10 mmHg	1.18	(1.07, 1.31)	.002	1.06	(0.94, 1.20)	.36	-	-
Diastolic blood pressure, 10 mmHg	1.20	(1.02, 1.41)	.03	1.09	(0.91, 1.31)	.33	-	-
eGFR, 10 ml/min/1.73 m2	0.41	(0.35, 0.48)	<.001	0.47	(0.40, 0.56)	<.001	-	-
Related to recipient	0.86	(0.63, 1.17)	.33	1.26	(0.89, 1.78)	.19	-	-
Nephron size and number
Glomerular volume, standard deviation (SD)	0.95	(0.81, 1.11)	.49	0.96	(0.80, 1.15)	.65	0.93	(0.77, 1.13)	.46
Cortex volume per glomerulus, SD	1.13	(0.96, 1.32)	.14	1.05	(0.89, 1.25)	.57	1.02	(0.85, 1.23)	.82
Mean tubular cross-sectional area, SD	1.13	(0.97, 1.32)	.11	1.11	(0.93, 1.32)	.26	1.09	(0.90, 1.32)	.40
Nephron number <5th percentile b	1.77	(0.96, 3.25)	.07	2.10	(1.07, 4.13)	.03	1.79	(0.89, 3.64)	.10
Nephrosclerosis
Globally sclerotic glomeruli >95th percentile b	1.45	(0.77, 2.74)	.25	1.62	(0.80, 3.26)	.18	1.50	(0.69, 3.27)	.31
Percentage IF/TA >95th percentile b	1.20	(0.70, 2.06)	.51	1.58	(0.88, 2.85)	.13	1.80	(0.95, 3.42)	.07
Number of IF/TA foci >95th percentile b	2.00	(0.99, 4.04)	.05	1.49	(0.68, 3.27)	.32	1.59	(0.68, 3.72)	.28
Artery luminal stenosis >95th percentile b	0.66	(0.31, 1.41)	.28	0.79	(0.33, 1.89)	.58	0.69	(0.27, 1.74)	.43
Any arteriolar hyalinosis	1.37	(0.83, 2.26)	.21	0.89	(0.51, 1.54)	.67	0.96	(0.53, 1.73)	.88

^a.Adjusted for time from donation, age, gender, and eGFR

^b.Age specific thresholds in Supplemental Table 1 and Supplemental Table 2

Table 5.

Predictors of residual eGFR (post-donation eGFR/ pre-donation eGFR × 100%) among 673 donors with follow-up serum creatinine levels. (Residual eGFR=74.8±14.9)

	Unadjusted			Adjusted for age, gender, and follow up time			Further adjusted for significant clinical predictors a
Clinical Predictors	% diff.	95% CI		% diff.	95% CI	P	% diff.	95% CI	P
Time since donation, 5 year	2.57	(0.77, 4.37)	.005	-	-	-	-	-	-
Age, 10 years	−2.31	(−3.25, −1.37)	<.001	-	-	-	-	-	-
Male	−1.65	(−4.00, 0.71)	.17	-	-	-	-	-	-
Body mass index, 5 kg/m2	−0.44	(−1.57, 0.69)	.44	−0.4	(−1.53, 0.72)	.48	-	-	-
Smoker at donor evaluation	4.92	(1.30, 8.54)	.008	3.81	(0.23, 7.40)	.04	-	-	-
Hypertension	−2.17	(−4.96, 0.62)	.13	−0.19	(−3.08, 2.70)	.90	-	-	-
Systolic blood pressure, 10 mmHg	−0.46	(−1.21, 0.29)	.23	−0.28	(−1.10, 0.53)	.50	-	-	-
Diastolic blood pressure, 10 mmHg	−0.67	(−1.85, 0.52)	.27	−0.27	(−1.46, 0.91)	.65	-	-	-
eGFR, 10 ml/min/1.73 m2	−1.90	(−2.65, −1.15)	<.001	−3.72	(−4.54, −2.89)	<.001	-	-	-
Related to recipient	0.20	(−2.06, 2.45)	.86	−1.04	(−3.30, 1.22)	.36	-	-	-
Nephron size and number
Glomerular volume, standard deviation (SD)	0.15	(−1.01, 1.30)	.80	0.09	(−1.09, 1.26)	.89	−0.25	(−1.36, 0.86)	.66
Cortex volume per glomerulus, SD	−0.84	(−2.00, 0.31)	.15	−0.72	(−1.88, 0.44)	.22	−1.01	(−2.10, 0.08)	.07
Mean tubular cross-sectional area, SD	−0.50	(−1.63, 0.64)	.39	−0.50	(−1.66, 0.65)	.39	−0.84	(−1.93, 0.25)	.13
Nephron number <5th percentile b	−3.52	(−8.02, 0.97)	.12	−4.16	(−8.58, 0.27)	.07	−6.07	(−10.24, −1.89)	.004
Nephrosclerosis
Globally sclerotic glomeruli >95th percentile b	−2.60	(−7.31, 2.10)	.28	−2.66	(−7.28, 1.96)	.26	−3.51	(−7.87, 0.86)	.11
Percentage IF/TA >95th percentile b	−2.34	(−6.35, 1.66)	.25	−2.92	(−6.86, 1.01)	.14	−2.98	(−6.69, 0.73)	.11
Number of IF/TA foci >95th percentile b	−4.02	(−9.16, 1.11)	.12	−2.37	(−7.48, 2.74)	.36	−2.58	(−7.39, 2.24)	.29
Artery luminal stenosis >95th percentile b	4.37	(−0.65, 9.38)	.09	3.60	(−1.43, 8.63)	.16	3.25	(−1.70, 8.21)	.19
Any arteriolar hyalinosis	−1.41	(−5.01, 2.19)	.44	0.20	(−3.38, 3.78)	.91	0.19	(−3.21, 3.59)	.91

^a.Adjusted for time from donation, age, gender, and eGFR

^b.Age specific thresholds in Supplemental Table 1 and Supplemental Table 2

3.4. Predictors of self-reported proteinuria

Of the surveyed donors, 40 of 786 (5.1%) reported being diagnosed with abnormal proteinuria during follow-up. Clinical predictors of abnormal self-reported proteinuria were younger age, male gender, pre-donation hypertension, and higher diastolic blood pressure (Table 6). Larger glomerular volume predicted self-reported proteinuria and this association remained evident after adjusting for all clinical characteristics. Low nephron number and nephrosclerosis did not predict proteinuria before or after adjusting for clinical characteristics (Table 6).

Table 6.

Predictors of self-reported proteinuria that occurred in 40 of 786 (5.1%) donors.^d

	Unadjusted			Adjusted for age, gender, and follow up time			Further adjusted for significant clinical predictors a
Clinical Predictors	OR	95% CI	P	OR	95% CI	P	OR	95% CI	P
Time since donation, 5 year	1.34	(0.79, 2.25)	.27	-	-	-	-	-	-
Age, 10 years	0.74	(0.57, 0.97)	.03	-	-	-	-	-	-
Male	1.96	(1.03, 3.71)	.04	-	-	-	-	-	-
Body mass index, 5 kg/m2	1.28	(0.96, 1.71)	.09	1.25	(0.93, 1.68)	.13	-	-	-
Smoker at donor evaluation	1.45	(0.59, 3.56)	.42	1.32	(0.53, 3.26)	.55	-	-	-
Hypertension	1.66	(0.81, 3.40)	.17	2.50	(1.12, 5.55)	.02	-	-	-
Systolic blood pressure, 10 mmHg	1.15	(0.94, 1.41)	.17	1.21	(0.96, 1.53)	.10	-	-	-
Diastolic blood pressure, 10 mmHg	1.36	(0.98, 1.87)	.06	1.42	(1.02, 1.97)	.04	-	-	-
eGFR, 10 ml/min/1.73 m2	0.99	(0.80, 1.22)	.90	0.83	(0.65, 1.07)	.16	-	-	-
Related to recipient	1.54	(0.80, 2.95)	.19	1.29	(0.66, 2.52)	.46	-	-	-
Nephron size and number
Glomerular volume, standard deviation (SD)	1.55	(1.20, 2.00)	<.001	1.47	(1.13, 1.92)	.005	1.42	(1.08, 1.86)	.01
Cortex volume per glomerulus, SD	1.23	(0.95, 1.58)	.12	1.25	(0.95, 1.63)	.11	1.21	(0.93, 1.59)	.15
Mean tubular cross-sectional area, SD	1.17	(0.86, 1.59)	.32	1.13	(0.83, 1.55)	.44	1.09	(0.79, 1.49)	.61
Nephron number <5th percentile b	1.14	(0.34, 3.87)	.83	1.21	(0.35, 4.14)	.76	1.24	(0.36, 4.29)	.73
Nephrosclerosis
Globally sclerotic glomeruli >95th percentile b	0.86	(0.20, 3.69)	.84	0.93	(0.21, 4.05)	.92	0.93	(0.21, 4.10)	.93
Percentage IF/TA >95th percentile b	0.89	(0.27, 2.99)	.86	0.87	(0.26, 2.91)	.82	0.84	(0.25, 2.85)	.78
Number of IF/TA foci >95th percentile b	<0.001 c	(0.00, 0.94)	.04	<0.001 c	(0.00, 1.15)	.07	<0.001 c	(0.00, 1.08)	.06
Artery luminal stenosis >95th percentile b	0.96	(0.23, 3.94)	.95	0.92	(0.22, 3.80)	.91	0.95	(0.23, 3.90)	.95
Any arteriolar hyalinosis	1.74	(0.69, 4.44)	.24	1.65	(0.63, 4.35)	.31	1.62	(0.61, 4.27)	.33

^a.Adjusted for time from donation, age, gender, hypertension and diastolic blood pressure

^b.Age thresholds in Supplemental Table 1 and Supplemental Table 2

^c.0 donors had both abnormal number of IF/TA foci and self-reported proteinuria at follow-up.

^dThere were 21 patients who did not answer the proteinuria query on the follow-up survey.

4. DISCUSSION

Clinical factors such as GFR, obesity, and blood pressure are carefully considered when evaluating the long-term risks with kidney donation. Despite this, subclinical microstructural features in the donated kidney at the time of transplantation are still predictive of developing CKD a decade after donation. Specifically, larger glomerular volume predicts an increased risk of eGFR <45 ml/min/1.73 m² or self-reported proteinuria and low nephron number predicts an increased risk of eGFR <45 ml/min/1.73 m² or lower residual eGFR (i.e., a larger decline in eGFR). These risks are small and would probably not justify obtaining kidney biopsies prior to donation. However, they provide insights into the microstructural features in the kidney that are important to long-term kidney health among relatively healthy individuals.

Whether or not living kidney donation causes hypertension has been debated.^{25, 26} Nonetheless, hypertension is common after kidney donation.^{27, 28} We previously found arteriosclerosis to associate with concurrent hypertension in these donors¹⁵ and to predict new-onset hypertension at a mean of 4 months following donation.¹⁸ However, in this current study, arteriosclerosis did not predict new-onset hypertension at a mean 10.5 years after donation. This suggests that with the passage of time, other factors predominately determine the risk of hypertension after donation. Interestingly, while age did not predict hypertension, longer time since donation predicted hypertension. This may be related to the selection for good health at the time of donation for both younger and older donors. As others have found,²⁹ there was an increased risk of hypertension after donation associated with obesity. Larger glomeruli occur with a higher BMI, even in the BMI range of kidney donors.¹⁷ We found that several measures of larger nephron size predicted hypertension but not in models that further adjusted for BMI. This is consistent with larger nephron size being part of the pathway by which obesity contributes to the risk of hypertension.

Long after kidney donation, eGFR continues to rise in most donors,^{25, 30, 31} though some donors still develop CKD.⁹ We did not find nephrosclerosis at donation to predict a long-term risk of a low eGFR. While larger glomeruli associates with a high GFR in donors at the time of donation,¹⁴ we found larger glomeruli to predict a long-term risk of low eGFR (<45 ml/min/1.73 m²) after donation. A plausible explanation for this is that with further hyperfiltration after uni-nephrectomy, enlarged glomeruli may be more prone to collapse and sclerosis leading to lower eGFR. Alternatively, the development of lower eGFR with larger glomeruli may have occurred even without the nephrectomy. Importantly, this risk of eGFR <45 ml/min/1.73 m² with larger glomeruli persisted even after adjusting for baseline clinical characteristics including hypertension. A high proportion of donors had an eGFR<60 ml/min/1.73 m² at follow-up that was not predicted by any microstructural feature. An eGFR of 45–59 ml/min/1.73 m² may just be the direct effect of a nephrectomy rather than a kidney disease per se.

Low nephron number for age also associates with a lower residual eGFR and a low eGFR at follow-up. This was evident even after adjusting for baseline eGFR and other clinical predictors. In this healthy population, nephron number is reflective of nephron endowment and nephron loss from nephrosclerosis (global glomerulosclerosis in particular). Since nephrosclerosis measures did not predict a lower residual eGFR or low eGFR, low nephron endowment may be the primary contributor to this risk of a lower residual eGFR or a low eGFR. Birthweight correlates with nephron endowment,^{16, 32–36} but was not available in this cohort.

To our knowledge, only one other study looked at the microstructural predictors of long-term kidney function in donors after donation (310 donors with a median 6.2 years of follow-up).³⁷ This study found that IF/TA was a predictor of eGFR decline during follow-up. Consistent with this prior study, we did find IF/TA >95^th percentile for age trended in the same direction as in the prior study but was not statistically significant (p=.11).

There were potential limitations to this study, including reliance on a survey for outcomes. Previous studies have shown that with regards to hypertension, the concordance between a survey-reported diagnosis and a confirmed diagnosis of hypertension is high.^{38, 39} It is also reassuring that serum creatinine values on medical record review were well correlated with those on the surveys in the local subset. Another limitation in our study was the reliance on self-reporting for being diagnosed with abnormal proteinuria. There may have been under-detection of proteinuria due to lack of testing or lack of awareness of the proteinuria result even if testing was done. However, since donors and providers were unaware of the microstructural findings on the biopsy (pathology report did not include morphometry and was in the recipient’s medical record), a differential bias with respect to glomerular volume and the risk of self-reported proteinuria is unlikely. There were some baseline differences in the characteristics between those who responded to the survey versus non-responders and between those that reported a follow-up serum creatinine level and those that did not. A larger sample size with more events is needed adjust for more covariates in the models. The donors studied were predominately white, and further work is needed to determine if prediction of outcomes by kidney microstructural characteristics differs by race.

5. CONCLUSION

In summary, lower nephron number and larger glomerular volume were long-term predictors of CKD in kidney donors after donation, independent of other clinical predictors. Whether or not the donor nephrectomy itself contributes to this finding is unclear. Nephron number and glomerular volume are not typically reported by pathologists on biopsy reports. Further studies are needed to determine how these subclinical microstructural kidney features increase the long-term susceptibility for CKD in otherwise healthy adults. Ideally, less invasive biomarkers are needed to detect these microstructural features in order to make their evaluation feasible in more clinical settings.

Abbreviation Page

BMI

Body mass index

eGFR

Estimated glomerular filtration rate

GFR

Glomerular filtration rate

GSG

Globally sclerotic glomeruli

IF/TA

Interstitial Fibrosis/Tubular Atrophy

SBP

Systolic blood pressure

DBP

Diastolic blood pressure

CKD

Chronic Kidney Disease

ESRD

End Stage Renal Disease

CT

Computed Tomography